Acupuncture promotes mTOR-independent autophagic clearance of aggregation-prone proteins in mouse brain

January 24, 2016

Abstract

Acupuncture has historically been practiced to treat medical disorders by mechanically stimulating specific acupoints with fine needles. Despite its well-documented efficacy, its biological basis remains largely elusive. In this study, we found that mechanical stimulation at the acupoint of Yanglingquan (GB34) promoted the autophagic clearance of α-synuclein (α-syn), a well known aggregation-prone protein closely related to Parkinson’s disease (PD), in the substantia nigra par compacta (SNpc) of the brain in a PD mouse model. We found the protein clearance arose from the activation of the autophagy-lysosome pathway (ALP) in a mammalian target of rapamycin (mTOR)-independent approach. Further, we observed the recovery in the activity of dopaminergic neurons in SNpc, and improvement in the motor function at the behavior level of PD mice. Whereas acupuncture and rapamycin, a chemical mTOR inhibitor, show comparable α-syn clearance and therapeutic effects in the PD mouse model, the latter adopts a distinctly different, mTOR-dependent, autophagy induction process. Due to this fundamental difference, acupuncture may circumvent adverse effects of the rapamycin treatment. The newly discovered connection between acupuncture and autophagy not only provides a new route to understanding the molecular mechanism of acupuncture but also sheds new light on cost-effective and safe therapy of neurodegenerative diseases.

Introduction

Aggregation-prone proteins, e.g. α-synuclein (α-syn), amyloid-β (aβ), Tau and polyglutamine-containing proteins (poly Q) are closely related to age-related neurodegeneration as seen in Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD)1,2,3. For example, PD, one of the most common neurodegenerative diseases (NDDs) in the elderly, is closely related with α-syn aggregation in brain2,3. It has been well established that cytosolic and misfolded proteins are degraded via the ubiquitin proteasome system (UPS) and the autophagy-lysosome pathway (ALP) under physiological conditions3,4,5. Previous studies in mice demonstrated that impairment of basal autophagy perturbs protein degradation in the central nervous system and causes NDDs6. Hence, clearance of aggregation-prone proteins by enhancing the autophagic level holds great promise in therapeutic treatment for NDDs3,5.

Macroautophagy (hereinafter called autophagy) is a highly conservative biological degradation pathway that plays essential roles in homeostasis, development and survival7. As a specific inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway, rapamycin has proven to be a potent inducer of autophagy and used for the treatment of certain diseases3,8. More specifically, it can attenuate toxicity of aggregate-prone proteins by activating ALP to clear protein aggregation in several types of NDDs2,8. However, rapamycin is known to have various adverse effects since it broadly affects many aspects of metabolism8. Hence, it is highly desirable to develop safer therapeutic approaches that specifically target downstream ALP in an mTOR-independent manner3.

Acupuncture is a treatment of traditional Chinese medicine (TCM) that mechanically stimulates discrete acupoints with fine needles. Acupuncture is practiced worldwide now, with accepted efficacies in treating many disorders and endorsement by the World Health Organization (WHO) and the US National Institutes of Health (NIH)9,10,11. However, the efficacy of acupuncture often raises considerable controversy in evidence-based medicine12, which largely arises from the lack of general mechanistic understanding of acupuncture. In this work, we aimed to study the positive roles of acupuncture for treating NDDs, which have been well documented for studies in experimental animals and patients13,14,15,16,17. Given that the nature of acupuncture is mechanical stimulation, and that autophagy is closely related with many physical factors, e.g. starvation, exercise and mechanical stress18,19, we were inspired to investigate the mechanism of acupuncture, the specificity of acupoints and PD treatment in a PD mouse model under the context of autophagic regulation.

Results

Acupuncture relieved the accumulation of α-synuclein in PD mice

We used a PD mouse model by treating C57BL/6 mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at a dose of 30 mg/kg/d for five consecutive days (the PD mice group, PG), which is a well-established animal model with PD-like neuropathology20,21,22,23. We also found that the expression of α-syn was significantly increased in the substantia nigra par compacta (SNpc) of the PG mouse brain (Fig. 1). Concomitantly, we observed PD-like pathological changes including motor dysfunction and dopamine (DA) neuron impairment in SNpc of PG mice. Stimulation of Yanglingquan (GB34), which is located at the depression anterior and inferior to the fibular head16, is traditionally used for the motor function treatment in TCM24. Several previous studies have shown that acupuncture at GB34 can protect the dopaminergic neuron against MPTP25,26,27,28. Hence, we selected GB34 as the acupoint in this study. Acupuncture stimulation was performed once a day for 12 consecutive days at the acupoint GB34 in the acupuncture group (AG) or at a control point that is located at the lateral side (by 3 mm) of the tail in the sham acupuncture group (SG) (Fig. 1a)25,26,27,28. Significantly, we found that the expression level of α-syn in SNpc was decreased by ~56% in AG, which remained nearly constant in SG (Fig. 1b). This remarkable difference in treating different points reflects the site-specificity of acupoints and acupuncture, which is consistent with the acupuncture theory in TCM29,30.

Acupuncture promoted the autophagic clearance of α-synuclein in an m-TOR-independent pathway

Having established that acupuncture suppresses α-syn expression in SNpc, we explored the relationship between the observed α-syn clearance and autophagy. We first examined the sub-cellular structures in SNpc of mouse brain by using transmission electron microscopy (TEM). Mice in PG showed significant changes in the structure and distribution of mitochondria and lysosome as compared with those in the saline group (Fig. 2 and Supplementary Fig. 2). The mitochondria had a substantial loss of matrix density, swelling and cristae disruption, and the number of lysosomes significantly decreased. Notably, we found the appearance of abundant autophagosomes, which are characteristic of double-membrane structures containing intact or partly degraded cytoplasmic material31. The accumulation of autophagosomes along with the lysosomal impairment in PG mice are consistent with previous studies23, which suggests the dysfunction of the ALP in these mice with PD27. Remarkably, the acupuncture treatment at GB34 largely restored the structures of mitochondria and lysosome along with significant reduction of the autophagosome accumulation in AG (Fig. 2). In contrast, mice in SG did not show apparent effects on the structure of mitochondrion and lysosome (Fig. 2). Hence, acupuncture at the specific acupoint GB34 exhibits apparent autophagy induction in the SNpc of mice.

We next analyzed the expression of ALP-related proteins to investigate the role of ALP in these processes. In ALP, microtubule-associated protein 1 light chain 3 II (LC3II) is a typical protein marker associated with the completed autophagosomes, and lysosome associated membrane protein 1 (LAMP1) is a lysosomal structural protein23,31. We first examined the expression levels of LC3II and LAMP1 in SNpc of PG mice. We found that LC3II was up-regulated, suggesting either the induction of autophagy or interruption of the fusion of autophagosome and lysosome31, and LAMP1 was down-regulated, suggesting the impairment of lysosome (Fig. 3a,b). Hence, it is the interruption of fusion of autophagosome and lysosome that leads to the accumulation of autophagosomes in SNpc of PG mice31. Significantly, compared with mice in PG, we observed ~37% decrease in the expression level of LC3II and ~20% in LAMP1 on the 4th day in AG, along with over 50% clearance of α-syn in SNpc (Fig. 3a,b). These data suggest that acupuncture significantly rescues the ALP to enhance the clearance of autophagosomes and the degradation of α-syn. Double immunofluorescence examination was performed to locate lysosome with an anti-LAMP2 antibody and DA neurons with an anti-tyrosine hydroxylase (anti-TH) antibody, which further confirms the appearance of more lysosomes in SNpc DA neurons after the acupuncture treatment (Fig. 3c). We further note that the low expression of α-syn maintained till the 7th day in AG, suggesting the sustainable effects of acupuncture. Also of note, mice in SG did not show apparent ALP restoration or significant α-syn clearance, which coincides well with the structural analysis in SNpc.

Rapamycin enhanced α-syn clearance in an m-TOR-dependent pathway

Since LC3II and LAMP1 are both downstream ALP proteins, we further examined the acupuncture effects on the expression of upstream ALP proteins of p-mTOR, p-P70S6K and ULK1 (Fig. 4c–f). However, these upstream proteins were minimally affected by acupuncture, suggesting that its regulating effects on ALP are independent of mTOR32. We also compared acupuncture treatment with the treatment of an FDA-approved drug, rapamycin, which is a well known autophagic enhancer. The effects of rapamycin on downstream proteins (LC3II and LAMP1) in the rapamycin-treated group (RG) were generally similar to those of acupuncture (Fig. 4a and Supplementary Fig. 3c-e). Nevertheless, rapamycin could significantly modulate the expression of upstream ALP proteins including p-mTOR, p-P70S6K and ULK1 (Fig. 4c–f), suggesting that rapamycin not only restored the lysosomal level but also induced autophagy by inhibiting the m-TOR pathway.

Acupuncture rescued the PD symptoms

Having substantiated the acupuncture-induced autophagic clearance of α-syn, we next examined the therapeutic effect of acupuncture on the motor function, the number and the activity of DA neurons in mice of PG. PG mice were slow in movement with postural instability. In order to quantify their motor function, we measured the overall rod performance (ORP) score using a rota rod instrument33 (Fig. 5b). The ORP scores of mice in PG (1527.2 ± 238.6) were nearly half of those in the saline group (3401.6 ± 426.4). Remarkably, AG mice exhibited ~80% increase in the ORP scores (2728.1 ± 426.4), whereas SG ones did not show significant change (Fig. 5c). The observations on the behavior improvement are generally consistent with previous reported efficacies in acupuncture treatment for PD animals and patients13,14,15,16,17. We also note that PG mice showed sparse and dull hair coat, whereas that of AG mice was thick and shiny.

We further examined the number of TH-positive neurons and the level of DA, which reflect the number and the function of DA neurons, and the c-fos and synaptophysin proteins that reflect the activity of dopaminergic neuron. In PG mice, the number of TH-positive neurons in the SNpc and striatum respectively decreased by ~57% and ~78%, along with the reduction of DA concentration (PG: 2.72 ± 0.91 ng mg−1tissue vs. saline: 7.72 ± 0.43 ng mg−1 tissue), whereas the c-fos expression increased by ~43% (Fig. 5a,d–h). After the acupuncture treatment at GB34, the number and function of DA neurons restored significantly in AG, with the density of TH-positive neurons in the SNpc and striatum increased by ~38% and ~61%, respectively (Fig. 5a,d,e). The DA concentration in AG was increased to 3.90 ± 1.22 ng mg−1 tissue(Fig. 5h). The synaptophysin protein expression in AG increased about by ~40% as compared with that in PG mice (Fig. 5i–j), which further confirmed that acupuncture rescued the loss of DA neurons in PG mice and improved the release of DA neurotransmitter. We also note that such a neuroprotective effect was not found in SG.

To read more:

http://www.nature.com/articles/srep19714